Use of antibodies specific for peptide tags that have been previously engineered into polypeptides to purify those polypeptides by affinity chromatography and/or to coat those proteins on various solid supports is a widely used strategy and a number of peptide tags and their associated antibodies are already commercially available (GST(glutathion-S-transferase)-tag and GST-specific antibody, Invitrogen; FLAG™-tag and anti-FLAG™ antibodies, Sigma-aldrich Co, Strep-Tag™ and Anti-Strep-Tag™ antibodies, IBA . . . ).
The AviTag™ Technology
The AviTag™ sequence (U.S. Pat. Nos. 5,932,433, 5,874,239 & 5,723,584) is a unique peptide, just 15 residues long, that is recognized by biotin ligase (Schatz P. J., 1993). In the presence of ATP, the ligase specifically attaches biotin to the lysine residue in this sequence. Using vectors, the AviTag™ can be genetically fused to a much bigger polypeptide. This feature effectively allows any polypeptide that has been cloned to be tagged with a biotin molecule.
The originality of the AviTag™ is that this peptide can be biotinylated by the E. Coli enzyme BirA. Thus, polypeptides containing this biotinylated peptide either at their NH2 or at their COOH terminus can interact with very strong affinity with streptavidine and can be multimerized or attached to streptavidine-coated surfaces (Altman et al, Science, 1996; Bodinier et al, Nature Medicine, 2000; Rabu et al, J. Biol. Chem., 2005). Moreover, in vivo or in vitro biotinylation of polypeptides is possible.
The use of AviTag™ is growing rapidly in various industrial and medical applications that demand an efficient and robust immobilization technology, including biosensors, diagnostics, proximity assays and drug screening.
Cell Sorting Technology:
One of the possible uses of AviTag™ is its engineering into MHC heavy chains to form tagged MHC/peptide complexes and to further make tetramers of MHC/peptide complexes with streptavidin. These MHC/peptide tetramers specifically bind to T lymphocytes bearing the corresponding T cell receptors (TCR) (Altman et al, Science, 1996, patent WO96/26962) and are now widely used to monitor specific T cell responses in man and in animal models.
Similar biotinylated MHC/peptides complexes coated on streptavidin magnetic beads have been used to design a very efficient sorting procedure to isolate T lymphocytes specific for these complexes (Bodinier et al, Nature Medicine, 2000 and patent WO 0118053). However, biotin-streptavidin interaction is so strong (Kd=10−15 M) that it is almost irreversible and so, once bound to streptavidin-coated surfaces polypeptides containing the biotinylated AviTag™ cannot be detached. This has two major consequences:                Biotinylated polypeptides cannot be purified on streptavidine columns because elution is almost impossible;        When biotinylated polypeptides are coated on beads to sort specific cells bearing the corresponding ligands on their surface, the beads may remain bound to the cells for long periods of time and thus trigger transduction events that may have deleterious effects on the selected target cells.        
This second point has been well documented in recent publications in the case of the induction of apoptosis of specific T lymphocytes following incubation with MHC/peptides multimers. It was shown that incubation with MHC/peptides complexes of high valence containing short linkers induced rapid apoptosis of T lymphocytes in culture (Cebecauer et al, J. Immunol., 2005). This apoptosis was due to prolonged activation of these T lymphocytes by persistent cross-linking of their TCR.
A number of technical solutions have been proposed to increase the dissociation of MHC/peptide multimers and thus reduce apoptosis of sorted T lymphocytes. This was performed either by engineering a new short tag with reduced affinity for streptavidin into the MHC heavy chain (Strep-Tag™) and sort with magnetic beads coated with a modified streptavidin (Streptamers™, IBA) or by using desthiobiotin instead of biotin to make reversible MHC/peptide tetramers and then sort labelled cells by fluorescence-activated cell-sorting (Guillaume et al, J. Immunol. 2006). Both methods require the addition of an excess of free biotin to compete out the tagged MHC/peptide complexes from streptavidine and thus dissociate MHC/peptide multimers from the sorted cells.
Thus, there is still an important need for a tool which enables to reversibly attach a polypeptide bearing the AviTag™ on a solid support.